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Abstract

Objective— We have previously demonstrated that ischemic cardiomyopathy is associated with selective impairment of progenitor cell function in the bone marrow and in the peripheral blood, which may contribute to an unfavorable left ventricular remodeling process.

Methods and Results— With this study, we intended to identify the influence of telomere length on bone marrow functionality in 50 patients with coronary artery disease (CAD) and previous myocardial infarction. Mean telomere length (mTL) was measured simultaneously in peripheral blood leukocytes and mononuclear bone marrow cells (BMC), using the flow-FISH method. Telomere erosion already occurred at the bone marrow level, whereby age (39 bp/yr, P=0.025) and the number of affected vessels (434 bp/vessel, P=0.029) were the only independent predictors. Lymphocytes demonstrated significant TL shortening between BMCs and peripheral blood in CAD patients (−1011±897 bp) as opposed to an increase in a young control group (+235±459 bp, P<0.001). SDF- and VEGF-specific migration of BMCs correlated with mTL of lymphocytes (r=0.42, P<0.001) and was significantly reduced in CAD patients. Finally, the telomere length difference between granulocytes and lymphocytes was the most determinant for telomere-associated bone marrow impairment (P<0.001).

Conclusion— In patients with CAD, telomere shortening of BMCs is dependent on both age and the extent of CAD and correlates with bone marrow cell functionality.

Telomerase is a DNA polymerase that elongates the ends of chromosomes, which are formed by TTAGGG DNA repeats called telomeres.1–5 Telomeres are widely regarded as the internal biological clock of a living organism, and shorten by a few base pairs with every cell division.6 In addition, they are also crucial to maintaining chromosomal integrity.7–9 Overexpression or endogenously active telomerase in stem cells can counteract replicative telomere shortening.10 In vitro, telomere shortening can be accelerated by either loss of telomere capping proteins (ie, TRF2), oxidative stress, or DNA damaging noxes, each leading to premature senescence.7,8,11–18 Age-related pathologies with short telomeres include infertility, impaired wound-healing, and liver cirrhosis.3,19 Individuals with age-corrected shortage of leukocyte telomeres have a significantly higher risk of developing coronary heart disease or of dying from cardiovascular and infectious disease.20–24

van der Harst et al have recently shown that telomere length is shorter in patients with congestive heart failure (CHF) compared with age-matched control patients.19 Telomere shortening was also related to the severity of disease. We have shown previously that ischemic cardiomyopathy is associated with selective impairment of progenitor cell function in the bone marrow and in the peripheral blood, which may contribute to an unfavorable left ventricular (LV) remodeling process.25

Because we also observed a correlation with progenitor cell function and the overall cardiovascular risk factor load, whereas van der Harst observed a relationship with telomere length reduction and the extent of atherosclerotic disease manifestation, we hypothesized a link between telomere length and bone marrow function. The goal of this study was to identify predictors of bone marrow functionality associated with telomere biology in patients with ischemic cardiomyopathy.

Methods

Study Populations

For comparison of telomere length between bone marrow and peripheral blood, we simultaneously obtained peripheral blood and bone marrow samples from 50 CAD patients, 7 age-matched DCM patients, and 10 young and healthy volunteers after obtaining informed consent. For details please see the supplemental materials, available online at http://atvb.ahajournals.org.

Measurement of Telomere Length (Flow-FISH)

Telomere length measurements were carried out as previously described.27 In brief, each sample, containing 100 000 bovine thymocytes as an internal standard and 200 000 cells from the patient, was resuspended in a hybridization mixture containing telomere-specific N-terminal fluorescein isothiocyanate (FITC)-conjugated (C3TA2)3 PNA probe, washed, and counterstained with propidium iodide before analyzing by fluorescence-activated-cell sorter (FACS) analysis. For details please see the supplemental materials.

Assessment of Migratory Capacity of Bone Marrow Mononuclear Cells

A total of 1×106 BMCs were resuspended in serum-free medium and placed in the upper chamber of a modified Boyden chamber filled with matrigel. Endothelial basal medium supplemented with 20% fetal calf serum and recombinant human SDF or VEGF were added to the lower chamber for migration. After 24 hours of incubation, VEGF- and SDF-specific transmigrated cells were counted. For details please see the supplemental materials.

Statistics

Statistical analysis was performed with the software package JMP 6.03 for Macintosh. All correlations were calculated according to Spearmans coefficient. Comparison of 2 groups was calculated using either Fisher exact test, chi-squared tests, or unpaired t test. If the variances differed significantly among 2 groups we used Welch test. For multiple regression analysis the JMP Fit Model tool was used. For comparison of correlating r’s Steiger and Fishers Z test Dr Calvin P. Garbins software (FZT.exe) was used (www.class.unl.edu/psycrs/statpage/comp.html).

Results

Telomere Length of Mononuclear Bone Marrow Cells Depends on Age and Extent of Atherosclerotic Disease Manifestation

Several recently published studies have observed telomere length shortening in leukocytes from patients with coronary artery disease when compared to age-matched controls.19,24,28 It is not known, though, whether these differences in mean telomere length (mTL) are already preceded by changes in telomere length of bone marrow progenitors. Therefore, we first determined mTL of myeloid bone marrow cells in 50 patients with previous myocardial infarction (CAD; median age 62.6 years, median ejection fraction 35%, Table 1) using a flow cytometric approach after in situ hybridization of cells with a telomere specific (C3TA2) probe (Flow-FISH).29–31 Seven patients with nonischemic dilated cardiomyopathy (DCM; median age 63.1 years, median ejection fraction 33%) and 10 healthy and young adults (median age 27.8 years) served as control groups. NT-proBNP in DCM patients was twice as high as in the CAD group (1852 versus 999 pg/mL, n.s.; Table 1). As expected, ejection fraction correlated inversely with NT-proBNP serum levels (Spearman rank correlation r=0.537; P=0.018, data not shown). CAD patients had accumulated significantly more pack-years and were treated more often with statins (87 versus 29%, P<0.05; Table 1). All other clinical variables did not differ between these 2 groups.

Figure 1. A, Correlation of age and mean telomere length (mTL) in patients from the bone marrow study population (n=57, • CAD, ♦ DCM). Linear regression curve of the mean is shown. The indicated slope refers to the CAD population. B, Number of atherosclerotic disease manifestations and mean mTL from myeloid BMCs (n=57).

Having shown the presence of telomere shortening at the level of bone marrow residing progenitor cells, we next wanted to know whether mTL of bone marrow cells can be estimated from peripheral blood cell mTL in patients with CAD. Using the Flow-FISH method, we were able to differentiate between granulocytes and lymphocytes. Telomere length of peripheral blood granulocytes did reflect mTL of the BMC population extremely well (Spearman rank correlation r=0.91, P<0.001; Figure 2A). To a lesser degree, we also found mTL of lymphocytes to correlate well with BMC mTL (Spearman rank correlation r=0.75, P<0.001; Figure 2B). To test for comparison of correlated r’s we used Fishers Z test (z=2.97) and Hotelling t/Steiger’s Z test (z=4.15). Both tests therefore rendered a significant statistical difference (P<0.01) between r’s, indicating a stronger correlation of granulocyte versus lymphocyte mTL with the BMC population.

To investigate a potential cause behind the differing degree of correlation among granulocytes and lymphocytes, we calculated telomere length bp differences between BMCs and peripheral blood cells seperately in each study group. Granulocytes from young controls as well as older CAD and DCM patients showed similar rates of telomere length loss when compared to mTL of myeloid BMCs (−394±332 bp versus −543±614 bp versus −461±514 bp, n.s.; Figure 2C), attributed to proliferation of their hematopoetic precursors. Assuming that 50 to 100 bp are lost at the telomere end during each step of replication, this would correlate with 4 to 8 rounds of cell division. In contrast to granulocytes, lymphocyte telomeres demonstrated significant shortening in CAD and DCM patients as opposed to an increase in the young control group (−1011±897 bp versus −789±609 versus +235±459 bp, P<0.001; Figure 2D).

Telomere Length Determines Functional Capacity of BMCs

Finally, we investigated whether telomere length has an impact on the functional capacity of BMCs. Previous studies documented that the migratory capacity of BMCs toward cytokines determines the capacity of these cells to improve neovascularization after ischemia in an experimental hind limb ischemia model and in a clinical pilot trial.32,33 In accordance with previous studies, the migratory capacity of mononuclear bone marrow cells to VEGF (85±58 cells per high power field versus 153±105, P<0.01; Figure 3A) and SDF (144±72 versus 209±123, P=0.02; Figure 3B) was significantly reduced in the CAD group. Interestingly, VEGF-specific migration of mononuclear bone marrow cells correlated well with the absolute mTL of lymphocytes (Spearman rank correlation r=0.42, P<0.0001; Figure 3C). In contrast, VEGF-specific migration did not show a significant correlation with the absolute mTL of granulocytes (r=0.20, P=0.10) or BMCs (r=0.16, P=0.21). Univariate analysis revealed further parameters correlating inversely with BMC function, such as glucose serum levels, presence of CAD/DCM, age, risk factor load, and the difference in telomere base pairs between the granulocyte and lymphocyte cell population (ΔTELGr-Ly). Multivariate linear regression analysis revealed only ΔTELGr-Ly as an independent predictor of VEGF-specific BMC migration (Table 3). A more detailed analysis of ΔTELGr-Ly showed its dependence from BMC telomere length (P=0.017) and the cardiovascular risk load (P=0.007; supplemental Table I), suggesting that telomere length shortening above average, particularly in the lymphocyte population, may reflect altered hematopoetic cell function in patients with CAD. Accumulation of cardiovascular risk factors seemed to facilitate telomere shortening.

Figure 3. VEGF- (A) and SDF- (B) specific migration of BMCs in young healthy controls (n=10) and CAD (n=50) patients is shown (Cells per high power field were counted). P indicates the statistical significance against the control group. C, Correlation of lymphocyte mTL and VEGF-specific migration of BMCs (n=67).

Telomere Length Gap Between Granulocytes and Lymphocytes Is Increased in Patients With Previous Myocardial Infarction

To validate the data from our bone marrow study population concerning ΔTELGr-Ly, we analyzed blood samples from 63 CAD patients (mean age 65.4 years, range 60 to 76) and compared them to 23 age-matched healthy controls (mean age 65.2 years, range 57 to 77; supplemental Table I). Clinical characteristics of the 63 CAD patients were almost identical to the bone marrow study population and we included only clinically stable patients with a healed myocardial infarction, resulting in a mean left ventricular ejection fraction (LVEF) of 31±9%. More than 60% of patients were NYHA class I and II and 89% of patients were on statin therapy. Regarding the difference in base pairs between the granulocyte and lymphocyte cell population (ΔTELGr-Ly), we found a significant increase from 742±396 bp in healthy controls to 1055±570 bp in CAD patients (P=0.018, supplemental Figure II). The telomere gap correlated only with HDL cholesterol (r=−0.322, P=0.03). However, given the number of variables tested, this probability value should be interpreted with caution. No correlation was found for the other CAD risk factors LDL cholesterol, smoking, arterial hypertension, and diabetes (HbA1c).

Discussion

Telomere Shortening and Bone Marrow Function

Results from our previous study showed that patients with chronic postinfarction heart failure show functional exhaustion of their hematopoietic progenitor cell pool in the bone marrow niche, whereas the number of hematopoietic progenitors does not seem to be reduced in the bone marrow.25 The functional impairment of hematopoetic progenitor cells was mirrored by a reduced migratory capacity of progenitor cells mobilized into the blood. We can demonstrate now that accelerated shortening of peripheral blood lymphocytes, here reflected by an increase in the telomere gap ΔTELGr-Ly, was the only independent predictor of VEGF-specific migratory function of bone marrow mononuclear cells. During myocardial ischemia, angiogenic cytokines such as VEGF and SDF are released into the blood.34 Enhanced migratory capacity of bone marrow–derived progenitor cells, which contain specific receptors for VEGF and SDF on their surface, has been shown to improve neovascularization in patients after myocardial infarction as well as in the animal-model.32,33 Therefore, lack of telomere attrition appears to be a beneficial factor for vascular repair.

Telomere Length in Mononuclear Bone Marrow Cells

Our findings, that telomere length from peripheral white blood cells in patients with CAD correlates very well with their bone marrow residing precursors, justifies the quantification of telomere length from peripheral blood cells as an indicator of stem cell divisions. Accordingly, age-dependent decline in telomere length of mononuclear bone marrow cells was found to be very similar compared to peripheral blood cells, suggesting an ageing process already in stem and progenitor cells. The question arises, which cell compartment in patients with CAD is the target for disease-related telomere erosion. Hematopoetic stem cell ageing is characterized by cell intrinsic alterations, affecting genes involved in myeloid and lymphoid specification and function in different ways.35 Nevertheless, in contrast to lymphoid cells production of myeloid cells is normal in aged mice.36 A recent study by Ju et al demonstrated that telomere dysfunction provokes defects of the hematopoietic environment that impair lymphopoiesis but increase myeloid proliferation in aging telomerase knockout Terc−/− mice.37

Unlike in germ cells, replication of hematopoetic stem cells leads to telomere shortening and—in the long term—to the subsequent exhaustion of proliferative capacity. In our study we found that intraindividual telomere shortening between myeloid precursors and granulocytes was almost identical in all groups tested, young healthy controls, and older patients with previous myocardial infarction. This finding is not surprising, because granulocytes have a very limited life span of up to 8 days and lack the ability for further cell division. The average difference in mean telomere length of 400 base pairs between myeloid progenitor cells and granulocytes indicates 4 to 8 rounds of replication.

Lymphocytes and CAD

The lifespan of lymphocytes is very different from granulocytes, ranging between months and years,38 and T lymphocytes undergo further maturation in the thymus. In our study, mTL of lymphocytes decreased far quicker in CAD patients than in healthy controls. This could potentially be attributable to repeated activation of specific T cells or to cumulative oxidative damage during aging.39 The fact that memory T cells have shorter telomeres than their naïve counterparts suggests cellular proliferation is the primary stimulus for telomeric attrition.40 Because lymphocytes are the only peripheral blood cells expressing telomerase activity and therefore are able to maintain telomere length despite proliferation,41 a reduction in telomerase activity in specific lymphocyte subsets could have facilitated accelerated telomere erosion. With the exception of coronary plaque neutrophils, granulocytes are not known to express telomerase activity.42 Lymphocyte turnover (lymphocyte half life) and thymus output, which could have accounted for accelerated telomere erosion,38 were also not measured in our study. Hence we cannot make any assumptions, whether lymphocyte kinetics have an impact on the telomere gap in CAD patients. In healthy adults, a shift toward memory T-cells with ageing has already been published by our coauthors.30 Therefore, an increase in the CD28− subpopulation of CD4+ or CD8+ lymphocytes, containing much shorter telomeres than their CD28+ counterparts, or a shift from a naïve to a memory phenotype of T-lymphocytes in CAD patients could have contributed to an accelerated loss of telomere length in the total lymphocyte population. In conclusion, we would speculate that a persistent and increased inflammatory state in patients with severe atherosclerosis or congestive heart failure could result into an increased proliferation of lymphocytes, leading to a shift toward more mature populations and hence into shorter telomeres.

An association between telomere length and pump function has first been showed by Collerton and colleagues.43 Coronary artery disease, especially in the context of congestive heart failure, is accompanied by increased inflammation and leukocyte telomere shortening, as recently shown by van der Harst et al.19 This could lead to clonal expansion in CD4+ and CD8+ T-lymphocytes and thereby telomere erosion. Senescent CD28− T-lymphocytes in turn can also produce inflammatory cytokines, such as interferon (INF)-γ, interleukin (IL)-2, and IL-6,44 thus increasing inflammation and promoting a viscious circle.

Conclusions

In the study from van der Harst, patients with ischemic heart failure demonstrated shorter telomeres in leukocytes than those with nonischemic heart failure.19 In addition, patients with 3-vessel coronary heart disease had shorter telomeres than those with 1-vessel disease. These data argue in favor of a strong influence of the extent of atherosclerosis on telomere length in leukocytes. Brouilette et al show that the association of shorter telomeres with coronary heart disease is not a consequence of the disease.20 They conclude that shorter telomeres could contribute directly to the pathophysiology of atherosclerosis. van der Harst et al only performed a cross-sectional study and were unable to differentiate between cause and consequence.19 Brouilette et al measured telomeres before the onset of a myocardial infarction.20 However, the presence of (subclinical) atherosclerosis in these patients has not been excluded. Furthermore, neither groups measured telomeres before and after the onset of myocardial infarction/ischemic disease. Therefore we do not know what the effects of infarction/ischemic disease is on telomere length. Alternatively, short telomeres could simply reflect increased oxidative stress. Vascular smooth muscle cells (VSMCs) in fibrous caps express markers of senescence coinciding with markedly shorter telomeres compared with normal medial VSMCs.45 In vitro, oxidants induced premature senescence in VSMCs with accelerated telomere shortening and reduced telomerase activity.

There is no evidence that short telomeres precede heart failure, therefore increased inflammation might result into enhanced telomere attrition. The impact of oxidative stress and inflammation on telomere length seems to be differentially regulated between lymphoid and myeloid cell populations, even though both are affected. Interestingly, studies in late-generation mice doubly deficient in apoE and telomerase RNA suggest that short telomeres lead to impaired proliferation of both lymphocytes and macrophages, an important step in atherosclerosis development. The authors find a substantial reduction of atherosclerosis compared with control mice with intact telomerase, in spite of sustained hypercholesterolemia in response to the atherogenic diet, and conclude that telomere exhaustion resulting in replicative immunosenescence may also serve as a mechanism for restricting atheroma progression.46

Acknowledgments

The assistance of Carmen Schön and Tina Rasper is greatly appreciated.

Sources of Funding

This work was supported by grants of the Deutsche Forschungsgesellschaft (DFG Sp-502/4-2) and by the European Union (EVGN).

Disclosures

None.

Footnotes

Original received December 10, 2007; final version accepted January 31, 2008.